Effects of Some Charged Amino Acid Mutations on the Electron Self-Exchange Kinetics of Cytochrome b5

Two mutants of trypsin-solubilized bovine liver cytochrome b 5 (cyt b 5 ) have been prepared to elucidate the function of charged residues near the heme exposed edge in the electron self-exchange kinetics of cyt b 5 . The Glu44, Glu56, and Asp60 were mutated into alanines in mutant I (E44A/E56A/D60A) while Glu44, Glu48, Glu56, and Asp60 were mutated into alanines in mutant II (E44A/E48A/E56A/D60A). The electron self-exchange rates of cyt b 5 mutants I and II have been measured as a function of temperature and ionic strength using the one-dimensional 1 H NMR saturation transfer method. Under the condition of μ = 0.10 M and [cyt b 5 ] = 0.50 mM, the rate constant of the cyt b 5 mutant I is (4.0 ± 0.4) × 10 3 M - 1 s - 1 at 20 °C. The value rises to (7.2 ± 0.7) × 10 3 M - 1 s - 1 at 35 °C; data from 20 to 35 °C gave Δ H ⧧ = 6.8 ± 0.8 kcal mol - 1 and Δ S ⧧ = −19 ± 3 eu. The rate constant for cyt b 5 mutant II is (8.4 ± 0.7) × 10 3 M - 1 s - 1 at 20 °C with μ = 0.10 M and [cyt b 5 ] = 0.60 mM. The value rises to (17 ± 1) × 10 3 M - 1 s - 1 at 35 °C, Δ H ⧧ = 7.9 ± 0.9 kcal mol - 1, and Δ S ⧧ = −14 ± 4 eu. The rate constant for cyt b 5 mutants increases with increasing of ionic strength. The values of the rate constants of cyt b 5 mutants extrapolated to infinite ionic strength, k inf, are 2.8 × 10 5 and 1.1 × 10 5 M - 1 s - 1 for mutant I and mutant II, respectively. The protein dipole moment projections through the heme exposed edge for two mutants were obtained from a fit of ionic strength dependence of self-exchange rate constants using van Leeuwen's approach. The dipole moment projections were −220 D and −254 D for oxidized and reduced forms of cyt b 5 mutant I, and −134 D (oxidized) and −159 D (reduced) for cyt b 5 mutant II. In terms of Marcus theory, the reorganization energies of cyt b 5 mutant I and mutant II have been estimated to be 1.2 and 1.3 eV, respectively. The association constants for the electron self-exchange reactions of mutant I and mutant II are 0.33 and 1.5 M - 1, respectively. Based on above results, the recognition and electron self-exchange mechanism between oxidized and reduced states of cytochrome b 5 was discussed.